1. Field of the Invention
The present invention relates to the field of food science and engineering and, more particularly to dairy-based food products and their production including solutions to problems associated with lactose intolerance such as product processing methods and products produced by these methods.
2. Description of the Related Art
The nutritional problems associated with lactose, a disaccharide consisting of glucose and galactose, which in whole bovine milk constitutes 4.8 w/v % of the product, are well known. After weaning, humans may lose their ability to digest lactose due to a decrease in cellular production of the intestinal enzyme, lactase/β-galactosidase (β-D-galactoside galactohydrase, EC 3.2.1.23), which catalyzes the hydrolysis of the β(1-4) glycosidic bonds of lactose to obtain glucose and galactose monosaccharides at the brush border of the small intestine, allowing for uptake. The enzyme deficiency results in a variety of adverse health effects including gastrointestinal problems due to the inability of sugar to be hydrolyzed, and; consequently, absorbed. The undigested sugar then goes to the large intestine where it is fermented by microorganisms, producing acids and gas—resulting in flatulence, diarrhea, and cramping.
Estimates suggest that nearly 70% of the world's population is lactose intolerant, and that lactase deficiency corresponds to genetic populations originating from areas where milk and dairy are and were not staples in traditional diets, particularly affecting those of Asian and African decent. Likewise, those originating from areas with a history of milk consumption have adapted accordingly. Ethnically, the breakdown of lactose intolerance in the United States is: Caucasian-15%, Hispanic-53%, African-American-80%, and Asian-90%. There is, also, evidence that lactose intolerance increases with aging and loss of cellular production of the enzyme. In the United States, intolerance varies, with about one-third of the U.S. population having problems with lactose consumption. Lactose intolerance can result in the avoidance of dairy products, which account for over half of the daily calcium intake in the U.S. Insufficient calcium intake, in return, results in corresponding health issues, including osteoporosis. Current strategies for treating lactose maldigestion include lactase supplements, lactose-reduced dairy products, and avoidance of dairy products. Adverse nutritional and physical effects, as well as economic potential, have prompted research and opportunity in the utilization of lactose-reducing methods for the improvement and modification of milk and other dairy products.
Lactose, in food products can be reduced both prior to and after consumption of dairy. Methods of reduction include chemical hydrolysis, ultrafiltration, and utilization of external sources of the enzyme, β-galactosidase (lactase). In fluid milk products, the goal of lactose reduction is to hydrolyze the sugar between 70-100%. Less than 100% reduction is targeted because lactose has low sweetness relative to its monomers—glucose and galactose; which have a sweetness approximately 80% that of sucrose. The enhanced sweetness associated with hydrolysis is often unappealing to consumers, who have shown to significantly detect differences in sweetness at 80% lactose reduction. Chemical methods, which rely on acid hydrolysis of the sugar prior to consumption, are not useful in food products because of whole-food changes in sensory and nutritional loss that occur with the treatment. Ultrafiltration, as a processing step, has yet to find a market, because of energy and capital inputs, and the difficulty in separating lactose from similar sized molecules (i.e., vitamins). In the food industry, applications employing lactase to reduce lactose have been most successful.
Lactases are produced by microbial and in mammalian organisms, and are commercially available from yeast, mold, and bacterial sources. The common sources of commercially viable lactases are Kluyveromyces lactis, Candida pseudotropicalis, Aspergillus niger, and Aspergillus oryae. Mahoney makes the distinction that all lactases are β-galactosidases, while not all β-galactosidases are lactases because some plant cells and mammalian organs possess β-galactosidases that have little or no enzymatic activity on lactose. For food applications the diversity is reduced due to regulatory restrictions, being limited, as a direct food additive, to Kluyveromyces lactis and Candida pseudotropicalis, as well as Aspergillus oryzae and niger, which have GRAS approval. Lactases have been used to produce reduced lactose milk, improve the properties of ice cream in regards to crystallization, whipping, and viscosity properties, and for yoghurt to improve gelation, body, texture, and taste.
Lactase activity is affected by pH, temperatures, purity, and interaction with chemicals, which effects the application of the enzyme. Optimal pH and temperature varies with the source, modification, and method of preparation. Fungal lactases have an optimum pH range from 2.5-5.0, and yeast lactases range from 6.0-7.0. Yeast lactases are suited for fluid milk applications (pH 6.6-6.8), while fungal lactase are utilized in fermented diary products and for whey processing. The optimum temperature for fungal lactase is between 50-55° C., while yeast lactases range from 30-40° C., and are rapidly denatured over 40° C. Stability of lactase is affected by not only pH and temperature, but also interactions with other components, including divalent cations, lactose, and proteins found in milk. Mahoney and Wilder found that the half life of lactase from K. marxianus in milk was 20 times greater than in milk salts and 50 times greater than in phosphate buffer, presumably due to enhancement of hydrophobic interactions. Lactase purity is an important, though often overlooked, property of enzyme applications. Commercial sources of lactase have proteases present from incomplete purification of whole cell extracts that may degrade the product when in direct contact for a period of time. For milk applications, the presence of proteases can result in the production of undesirable texture and flavor changes. This factor is of special consideration for ultra high temperature (UHT) processed milk, which is processed to be stored for an extended time, and often have problems associated with proteases from sources other than lactase, and must be considered prior to application.
Applications that employ lactase to reduce lactose are useful for both fluid milk and whey processing. Lactose from whey, a byproduct of cheese manufacturing, can be hydrolyzed by the enzyme to produce sugars for use as prebiotics and sweeteners. Processing of whey with lactase provides not only nutritional advantages but also waste management improvements. Sweet whey, derived from the manufacturing of cheeses with rennet and acid whey, from manufacturing using acid coagulation, has, in the past been treated as a waste product. Whey components, particularly proteins, have found niche markets as nutritional supplements, edible films, and dried whey powder for emulsification and protein solubility in food applications. Whey powder, whey protein concentrate, and whey isolate, as ingredients for food applications, contain lactose. Reduction of lactose in these products provides not only advantages for lactose intolerant consumers, but also enhancements in food texture and consumer acceptability when using the ingredients because of the higher solubility of glucose and galactose compared to lactose and the decrease in lactose crystallization. The protein from whey can be removed by filtration making it even more suited for lactase activity. The permeate, which contains primarily lactose, vitamins and minerals, can be used for prebiotics, as syrup for utilized as a sweetener with increased solubility, or for animal feed. In a similar manner, permeate from skim milk is produced when microorganisms (and because of size similarities, proteins) are removed from the milk by ultra and microfiltration. The resulting permeate, which has a substantially reduced microbial load, is similar to that of sweet whey permeate. After processing, the fractionated portions can be added back to form a product with a lower bacterial count. Processing in this form may allow for the use of lactase, with minimal interferences from microorganisms, fat, and protein, to be used in the production of a lactose-reduced fluid milk product. Other applications of lactase to reduce lactose for consumption include: tablets that are taken prior to ingesting dairy, processing of fluid milk with lactase by the consumer, addition of the enzyme prior to or after pasteurization, and use of immobilized enzyme reactor.
Dosage forms, in which the enzyme is entrapped in a capsule and taken orally prior to the consumption of milk, can be used directly by the consumer. Doses must be taken in an appropriate time period to allow the enzyme to reach the intestine prior to consumption of dairy. Lactases added to milk are developed from yeast; however, those taken orally are from fungal sources to better accommodate the acidic conditions of the stomach. This method is, particularly, useful to consumers who eat-out and may ingest lactose. Lactose reduction by the consumer can, also, be achieved by the addition of a lactase tablets that is treated overnight at refrigeration temperatures.
The addition of lactase in milk prior to purchase by the consumer has been developed to provide convenience and decreased cost for the consumer while meeting a market demand and increasing profits for the producer. Lactase has been added prior or after pasteurization because of the susceptibility of yeast lactases to denaturation at processing temperatures used for batch, high temperature short time (HTST) and ultra high temperature (UHT) pasteurization, which occur at temperatures of 135° C. and higher. The addition of lactase prior to pasteurization has the advantage of producing a reduced lactose milk that can be processed, after reduction is completed, in a continuous manner that is identical to non-reduced milk. The disadvantages of this concept include an increase in holding time of the raw milk and vulnerability of the enzyme to proteases associated with the microbial load of the raw milk as well as ensuring completion of lactose reduction within the maximum 72 hour holding period in the plant.
Lactase addition after pasteurization is advantageous due to an increase in efficacy of the enzyme from a reduction in the microbial load and more time available for reaction. The disadvantage of this application is that a holding time at the enzyme's optimum temperature (30-35° C. for K. lactis) or lower is required after pasteurization to achieve the desired level of lactose reduction, after which the product goes through a second heat treatment to inactive biological contaminants that accompany commercial treatments of lactase and to stop lactose reduction if the desired level has been achieved. An alternative approach to the addition of lactase after pasteurization has been to inject a very small amount of yeast lactase through a sterile, 0.22 μm filter into UHT sterilized, packaged milk. The UHT product can be stored at room temperature, achieving complete hydrolysis in 7 to 10 days.
Lactase-Based Bioactive Systems
Immobilized Lactase for Industrial Reactors
Immobilized enzyme reactors have been utilized by the food industry to reduce the cost of enzymes during processing by binding the enzyme to an insoluble support. The advantages of immobilization include reuse, stability enhancement, and separation from the product. The enzyme may be conjugated to surface of the support by covalent bonding, ionic attachment, or hydrophobic interactions. The enzyme may, also, be incorporated into a polymer backbone during polymerization or physically entrapped in the bulk of a material. Lactase and whole cells contain lactase have been immobilized to a variety of supports by adsorption, entrapment, and covalent conjugation (see Table 1 and Table 2, infra). Though immobilized enzyme systems have many advantages, they are limited in practice by enzyme leakage, fouling, cost, enzyme activity, carrier stability, and microbial growth. Fouling can occur in both filtration membranes and in porous beads by constituents such as proteins found in milk, which limits the accessibility of substrate to the enzyme. For fluid milk, which has neutral pH, microbial growth is encouraged on immobilized supports. Sanitation of enzyme carriers is necessary to provide consumer safety, but is difficult and may lead to loss of enzymatic activity. For immobilized lactase, cleaning methods have been developed for immobilized A. oryzae (SEQ ID NO: 1) lactase using substituted diethylenetriamines.
TABLE 1Immobilization of lactaseTYPE OFMETHOD OF IM-SUPPORTLACTASEMOBILIZATIONPhenol-A. nigerAdsorption/70% hydrolysis offormaldehydeglutaraldehyde lactoseresinPorous aluminaA. nigerAdsorption/70-80% hydrolysisglutaraldehyde Phenol-A. nigerAdsorption/25 mg/g; 220 U/gformaldehydeglutaraldehyde resinEgg shellLactobacillusAdsorption/25% maximum bulgaricusglutaraldehyde activityPhenol-B. circulansAdsorption/225 U/g (wet)formaldehydeglutaraldehyde resinPhenol-A. nigerAdsorption/250 mg/g; 4000 U/gformaldehydeglutaraldehyde resinEgg white E. coliAdsorption/50% hydrolysis powderglutaraldehyde in 8 hrsFeather proteinA. nigerAdsorption/100 mg/g; 300 U/gglutaraldehyde proteinDEAE celluloseScopulariopsis Adsorption/1-2 U/g resinglutaraldehyde Phenol-A. nigerAdsorption/70-75% hydrolysisformaldehydeglutaraldehyde resinPolyacrylamideK. lactisHydrophobic bond70 U/gBrushiteE. coliAdsorptionN/ATritylagaroseE. coliHydrophobic bond75-90% relativeactivityNylon-E. coliCovalent-CMC or236.5 U/mg; PooracrylonitrilecarbodiimidestabilityAmino-K. fragilis/E. Covalent-3 U/g; 93 U/gcarbonilatedcolidiazo/celluloseglutaraldehydeCelluloseE. coliCovalent-benzo-109 mg/g, 4130 U/g;quinone/oxirane40 mg/g, 1780 U/gOxirane E. coliCovalent40 mg/g, 1300 U/g ofacryliccarrierSepharoseA. oryzaeCovalent63.9 U/mg(SEQ ID NO: 1) Oxirane poly-K. lactis/K.Covalent-oxiranepoor resultsacrylamidefragilisMn-Zn ferriteA. nigerCovalent-1 U/g; 16 U/gparticlessilanization/glutaraldhydePlexiglass-likeA. oryzaeCovalent80% hydrolysismaterial(SEQ ID NO:1)Ion exchangeA. oryzae (SEQCovalent-1000 U/g; 80%resinID NO: 1)glutaraldehydeconversion(purified)CelluloseK. lactisEntrapment30 mg/g; 22 U/gtriacetatePVOHK. lactisEntrapment100% hydrolysis in200 minPoly-LactobacillusEntrapmentMaximum 31% acrylamidebulgaricusactivityCollagenA. nigerEntrapment1680 U/gHollow fibreK. fragilisEntrapment40% hydrolysismembraneHollow fibreK. lactisEntrapment10 mg/930 cm squaremembrane
TABLE 2Covalent immobilization of lactaseTYPEMETHODSUPPORTOF LACTASEOF IMMOBILIZATIONNOTESPhenol-formaldehydeA. nigerAdsorption/glutaraldehyde70% hydrolysis of lactoseresinPorous aluminaA. nigerAdsorption/glutaraldehyde70-80% hydrolysisPhenol-formaldehydeA. nigerAdsorption/glutaraldehyde25 mg/g; 220 U/gresinEgg shellLactobacillusAdsorption/glutaraldehyde25% maximum activitybulgaricusPhenol-formaldehydeB. circulansAdsorption/glutaraldehyde225 U/g (wet)resinPhenol-formaldehydeA. nigerAdsorption/glutaraldehyde250 mg/g; 4000 U/gresinEgg white powderE. coliAdsorption/glutaraldehyde50% hydrolysis in 8 hrsFeather proteinA. nigerAdsorption/glutaraldehyde100 mg/g; 300 U/g proteinDEAE celluloseScopulariopsisAdsorption/glutaraldehyde1-2 U/g resinPhenol-formaldehydeA. nigerAdsorption/glutaraldehyde70-75% hydrolysisresinPolyacrylamideK. lactisHydrophobic bond70 U/gBrushiteE. coliAdsorptionN/ATritylagaroseE. coliHydrophobic bond75-90% relative activityNylon-acrylonitrileE. coliCovalent-CMC or carbodiimide236.5 U/mg; Poor stabilityAmino-carbonilatedK. fragilis/E. coliCovalent-diazo/glutaraldehyde3 U/g; 93 U/gcelluloseCelluloseE. coliCovalent-benzoquinone/oxirane109 mg/g, 4130 U/g; 40 mg/g,1780 U/gOxirane acrylicE. coliCovalent40 mg/g, 1300 U/g of carrierSepharoseA. oryzaeCovalent63.9 U/mgOxirane polyacrylamideK. lactis/K. fragilisCovalent-oxiranepoor resultsMn—Zn ferrite particlesA. nigerCovalent-silanization/glutaraldhyde1 U/g; 16 U/gPlexiglass-like materialA. oryzaeCovalent80% hydrolysisIon exchange resinA. oryzae (purified)Covalent-glutaraldehyde1000 U/g; 80% conversionCellulose triacetateK. lactisEntrapment30 mg/g; 22 U/gPVOHK. lactisEntrapment100% hydrolysis in 200 minPolyacrylamideLactobacillusEntrapmentMaximum 31% activitybulgaricusCollagenA. nigerEntrapment1680 U/gHollow fibre membraneK. fragilisEntrapment40% hydrolysisHollow fibre membraneK. lactisEntrapment10 mg/930 cm squareRETAINEDTYPE OFIMMOBILIZEDAMOUNTSUPPORTNATURE OF SUPPORTLACTASEACTVITYIMMOBILIZEDNOTESEupergit CCopolymer of methacrylamide,A. oryzae30% activityN,N′-methylen-bis(acrylamide)(SEQ ID NO: 1)and a monomer carrying oxiranegroups; Macroporous, ~150 umEupergit C-250LCopolymer of methacrylamide,B. circulans90% activity33 mg/gincrease ionicN,N′-methylen-bis(acrylamide)concentration,and a monomer carrying oxiraneincreased loadinggroups; Macroporous, ~250 um,and activity (uptohigher oxirane content1M); neutral andbasic coupling pHneeded; 10-24 hrcoupling time;immobilized onEuperit C withhigher loading andlower activityEupergit C-Copolymer of methacrylamide,K. lactis70-75%0.2 mg/glinked byglutaraldehye;N,N′-methylen-bis(acrylamide)carbohydrate areaEupergit C-epoxyand a monomer carrying oxiranedecreased productboronategroups; Macroporous, ~150 uminhibition; >20 hrsimmobilization timeSepabeadsMacroporous; polymethacrylateA. oryzaeno activitywith oxirane(SEQ ID NO: 1)Sepabeads-Amino-Macroporous; polymethacrylateA. oryzae3500 U/gUnclear (28-40 mg/g)>20 hrs reactionEpoxywith oxirane(SEQ ID NO: 1)time for completeimmobilizationGraphiteGraphite modified usingK. lactis8800 X0.63-1.30 mg/cmanhydrous methanol to introducedecrease insquarecarboxyl groupsactivityCottonTosylatedA. oryzae55%50 mg/g(SEQ ID NO: 1)Cotton fibers usingGlutaraldehyde cross-linkedA. oryzaeIn slurry,250 mg/gCotton wasPEI aggregates(SEQ ID NO: 1)high activity;immersed in PEI,Afterthen enzymecentrifugation,added whichlowaggregated theactivity;protein, all wasImmobilizedcrosslinkedactivity isunclearChitosanGlutaraldehyde cross-linkedA. oryzae~100%unclear(SEQ ID NO: 1)(0.1 mg/g)ChitosanGlutaraldehyde cross-linkedK. fragilis11-40%17-20 mg/gK. fragilis isnotoriouslyunstablePVOH-tosy sulfony chloride, cyanuricA. oryzae26-100%formaldehydechloride, benzoquinone(SEQ ID NO: 1)Salicylic acid,tosy-sulfonyl chloride, cyanuricA. oryzae14-28%resorcin,chloride, benzoquinone(SEQ ID NO: 1)formaldehydePhenol-glutaraldehydeA. niger200 umol/min/g5 mg/gformaldehyde resinof support;(plexiglass)40%Porous silicaPVC/silicaPEI/glutaraldehdyeA. oryzae90%1.9 mg/cm2ribbed increases(SEQ ID NO: 1)SA; rolled over likea capet around apollNylonGlycidyl methacrylateA. oryzae48-62%(diazotisation) glutaraldehyde(SEQ ID NO: 1)VmaxCelluloseEpichlorohydrinK. fragilis80%Unclearepoxy residueshave a slowreaction, and it hasbeen suggested byothers (Mateo) thatadsorption prior tocovalentmodification helpsSepharose 4BAgarose; 1-Cyano-4-K. lactis77-112%1-5.4 mg/mlcyanating protein(dimethylamino)-pyridiniumpacked supportsulfhydryl groups;tetrafluoroboratealso works onaminoControlled Porous3-aminopropyltriethoxysilaneK. fragilis90%Unclearlactase cultured inGlassactivated with glutaraldehydelabSiliva/aluminaaminopropyltriethoxysilaneK. fragilis50%activated with glutaraldehydeCPC/silicaaminopropyltriethoxysilaneK. lactis8-34%12.6-23 mg/mlactivated with glutaraldehydepacked supportGelatinglutaraldehyde or chromium (III)E. Coli22-25%acetateDEAE CelluloseglutaraldehydeScopulariopsis6x moreactivity thatduloliteCellulose beadsBenzoquinoneE. Coli23-83%13-109 mg/ghigh load reducedactivity butincreased stability.MW of E. Colilactase = 540,000Nylon/acryllmideglutaraldehyde and azideE. Coli156 U/gassumedsuggested poor2 mg/g basedsurface graftingon activitySilica•AluminaDiisocyanteA. niger100 folddecrease
Enzyme leakage and loss of activity over time has been demonstrated for entrapment and adsorption-based systems due to continuous processing. Covalent modification of the enzyme to or within a support has been explored as an approach to overcome the problem of enzyme leakage (see Table 2, supra). Different supports and methods of immobilization are developed so as to maintain/enhance enzyme activity, increase enzyme loading, lower the cost of immobilization, and optimize compatibility with a reactor design. The carrier (and support chemistry) must, also, be nontoxic, approved for food use, and, for covalent immobilization, have functional groups available for bioconjugation. For lactase immobilization, compromises are made to minimize or maximize these factors, and though some systems have been developed for industry, application to dairy processing has been allusive.
The nature of the support on enzyme activity is difficult to determine in part because of lack of free enzyme controls for comparison. Hydrophobic supports have been suggested to reduce the activity of the enzyme because of hydrophobic adsorption and complimentary unfoulding of the enzyme at the surface. Hydrophilic natural carriers including agarose, cellulose, dextran, alginate, gelatin, and collagen have been used with high activity retention. Hydrophilic carriers, though useful at the lab scale, are often not suited for industrial processing because of low mechanical rigidity (deformation), biodegradation, and swelling in aqueous solutions—leading to complications associated with pressure drops. Inorganic carriers including silica and glass have, also, shown promise with respect to activity retention. These supports have been used for industrial processes, but are limited by cost and pH stability.
Active Packaging
The growing trend in consumer demand for fresh, minimally processed, natural convenient foods with fewer additives along with changes in retail and distribution practices, has presented challenges to the food-packaging industry. In response to these trends and due the inherent limitation of traditional packaging systems to meet those demands has resulted in the development of active packaging applications. Active packaging involves interactions between a food, packaging material and the internal gaseous atmosphere. The goal of these systems is to, through the entrapment, absorption, or covalent linking of functional compounds to or within packaging materials, increase the quality and/or safety of the product after packaging. Such packaging changes the condition of the packaged food to extend shelf-life, or improve food safety or sensory attributes, while maintaining the quality of the packaged food. In this context, the polymer no longer has just passive properties as dictated by the chemical and physical structure, but also an active component that has been deliberately designed to serve a specific function in the food system
Enzymes that have been incorporated for active packaging include lysozyme, glucose oxidase, and nariginase. Lysozyme, an antimicrobial enzyme that is able to hydrolyze the β(1-4) linkages between N-acetylmuramic acid and N-acetylglucosamine, aiding in the break down of the cell wall of gram positive bacteria, has been immobilized on poly(vinyl alcohol) beads, nylon 6,6 pellets, and in cellulose triacetate films. Though all polymers demonstrated activity, the cellulose triacetate films showed the greatest efficiency, retaining 60% of their activity after 20 uses, and were showed to be inhibitory and bactericidal against Micrococcus lysodeikticus. Naringinase, which hydrolyzes the bitter compound naringin to naringenin and prunin, has been immobilized in cellulose acetate and cellulose triacetate polymers. The films showed a decrease in Km value, indicating an increase in the substrate affinity of the enzyme entrapped films. The films had an activity efficiency of up to 23% compared to the free enzyme at 7° C. Glucose oxidase, which converts glucose, oxygen, and water, to a glucono-delta-lactone and hydrogen peroxide, has been used in sachets and to perform as an oxygen scavenger. A difficulty with this system is that glucose must be available to serve as a reactant for the enzyme to perform as an oxygen scavenger, thus limiting its application thus far.
Lactose-reducing, heat sealable, packaging films have been by developed by lactase entrapment in ethylene(vinyl acetate) and covalently bound to poly(ethylene) with reduced activity upon immobilization. The enzyme has been attached to oxidized polyethylene films using a PEG intermediate and PEI-bound layer. Though the PEG-intermediate did not demonstrate activity, the PEI intermediate did retain measurable activity. A PEG-modified and native lactase has been entrapped in ethylene(vinyl acetate) films with both enzymes exhibiting a significant increase in Km and decrease in Vmax, but with detectable enzymatic activity. PharmaCal, Ltd has reported the use of an active packaging system using incorporated lactase that could reduce lactose 30-70% in 24-36 hours.
Loss of Enzyme Activity on Surfaces
The organization on an enzyme is that of a primary structure of covalently linked amino acids that form, along the sequence, globular, helical, and sheet folds based on hydrogen bonding (secondary structure). A tertiary structure is formed by the interactions of secondary structures—forming salt bridges, maximizing hydrophobic interactions, and satisfying requirements with the solvent. These interactions, also, form the basis of the catalytic cleft of the protein for enzymatic activity. The tertiary structure gives the enzyme a core of hydrophobic amino acids (phenylalanine, tyrosine, etc.) since exposure of these residues to a native hydrophilic environment would be unfavorable and a surface composed of hydrophilic and acid or basic amino acids because of their ability to hydrogen bond, and contains a bound water layer at the surface. In some cases, a quaternary structure is formed by noncovalent interactions of multiple subunits of same or different sizes.
The complexity of enzymes, though optimal for biological substrate specificity, presents challenges in the development of industrial applications. Enzymes have evolved to function under the conditions of their natural environment, and activity and stability are, subsequently, a reflection of that environment. The pH, temperature, intracellular or extracellular nature of the enzyme, protein concentration, molecular composition of the environment, salt concentration, water activity, substrate concentration, cellular function, and structure hierarchy may all influence the robustness of the enzyme and how it performs. Removing an enzyme from its native environment for use in a designed system changes the dynamics of the molecular equilibrium between the native and unfolded state of the enzyme. This equilibrium can be thermodynamically represented in the Gibbs Free Energy Equation (Equation 1, below)ΔG=ΔH−TΔS  Equation 1The equation can be expanded to include enthalpy (ΔH) terms for protein interactions, solvent interactions, and interactions of the two, as well as entropy (ΔS) terms for both the protein and the solvent (Equation 2, below)ΔG=ΔHsolvent/solvent+ΔHprotein/protein+ΔHprotein/solvent−TΔSsolvent−TΔSprotein  Equation 2For enzymes at a surface, the equation can be expanded further to include the insoluble surface (Equation 3, below)G=ΔHsolvent/solvent+ΔHprotein/protein+ΔHsurface/surface+ΔHprotein/solvent+ΔHprotein/surface+Hsurface/solvent−TΔSsolvent−TΔSprotein−TΔSsurface  Equation 3
The enthalpy terms account for inter- and intra-molecular interactions that can be changed to influence the enzyme state, including: bond lengths, van der Waals interactions, torsion angle, electrostatic interactions, and hydrogen bonding. The entropy terms indicate the order of the system, with the system favoring more disorder. With respect to an enzyme, dominating entropy will yield multiple, diverse confirmations of an enzyme, which, though energetically favorable, will produce an unfolded and inactive enzyme. Thermodynamic terms must; therefore, be sufficiently satisfied and work in such as way as to prevent unfolding of the catalyst.
Protein interactions at surfaces are studied at the liquid/liquid, liquid/gas, and liquid/solid interface. Proteins, because of their amphiphilic nature, are used in the formation of foams at the liquid/gas interface by denaturing of the tertiary structure to expose hydrophobic residues to the gaseous CO2 and liquid interface. Emulsions, similarly, incorporate proteins to stabilize solutions of oil and water constituents by reducing interfacial tensions and preventing coalescence by electrostatic repulsion. For immobilized enzymes, however, liquid/solid and solid/solid interactions are of importance, and enzyme activity can be lost or maintained due to the thermodynamic effects that occur at the surface interface between the enzyme and the carrier Amino acids that make-up the enzyme are capable of engaging in positive and negative electrostatic attraction and repulsion, disulfide bonding, and hydrophobic interactions—all of which may lower the free energy of the system when in contact with a surface. These groups, along with the hydrogen bonding of the bound water layer and the preferential hydration of a surface, impose restrictions on the nature of a solid interface in preventing loss of the native enzyme structure when the two are in contact.
Proteins with exposed hydrophobic groups have been shown to absorb tightly to hydrophobic surfaces due to lowering of the free energy in aqueous solution that occur from decreased exposed hydrophobic surface area when the two exposed groups come into contact. The hydrophobic interactions cause a dehydration of the protein surface, and the subsequent adsorption presents a shift in the tertiary or secondary structure of the protein. The molecule may then spread across the surface to further minimize the free energy. Both a small or large shift may cause a loss in enzymatic activity. Experiments seeking to characterize changes in enzyme secondary structure at the surface (compared to non-denaturing surfaces) have provided limited information since the shifts, though apparent, appear to be small compared to non-denaturing surface and random from enzyme to enzyme in regards to α-helix and β-sheet perturbations Consequently, changes in the secondary and tertiary structure are difficult to distinguish. Hydrophobic denaturation has been attributed to loss of lactase activity when the enzyme is bound to a porous polymethacrylate resin with oxirane groups.
Electrostatic groups on the surface of a support may lead to attraction or repulsion of ionic amino acids on the enzyme surface. When an enzyme is in contact with the surface, minimization or maximizing those interactions can lead to distortion of the protein and a loss of enzymatic activity. Binding of proteins to charge supports has been shown to occur even when the net charge on the enzyme is the same as the support because of ionizable amino acid side chains. The activity of lactase bound to an anionic support has been shown to be lower than activity when bound to a cationic support, suggesting a negative charge may alter the conformation of the enzyme. Carriers with surface ionic groups can also promote a pH shift in the microenviroment of the immobilized enzyme, which may change optimum catalytic conditions. These extremes in pH can alter protonation state of amino acids causing changes in hydrogen donor/acceptor characteristics, loss or gain or electrostatic repulsions, and changes in salt bridges.
In some instances hydrophilic surfaces have been shown to reduce enzymatic activity to a greater extent than a hydrophobic surface. This phenomenon is attributed to competitive hydrogen bonding at the interface or breaking of necessary salt bridges. The unique structure and chemical properties of water influences the interactions of a surface of an enzyme through hydrogen bond and dipole interactions. The hydrophilic surface changes the enzyme by competing and, ultimately, stripping the water layer from the enzyme shell, which is important from an enthalpic standpoint in maintaining tertiary structure (a hydrophobic surface may also strip the water surface by promoting dehydration—reducing the driving force to retain a hydrophobic residue inside the core).
Changing the chemical/physical nature of a carrier or distance from the surface can influence enzymatic activity, by limiting surface/protein interactions. The addition of the hydrophilic molecules to the surface of a hydrophobic material prevents proteins from adhering by reducing inter and intra-molecular hydrophobic interactions. Hydrophilic molecules, monomers, and polymers have been used to increase the number of functional groups on a surface or to provide a more reactive intermediate. Polyethylenimine, chitosan, polyacrylic acid, heparin, polyalylamine, alginate, and collagen have been grafted, either covalently or ionically, to facilitate protein loading or biocompatibility. The addition of the hydrophilic molecules to the surface of a hydrophobic material prevents proteins from adhering by reducing inter and intra-molecular hydrophobic interactions. Many of these polymers are, also, polyionic under conjugation conditions, which promotes ionic interaction with a charged protein. Initial ionic adsorption coupled with a means of covalent binding has been shown to promote protein loading on a material. A layer-by-layer approach to immobilizing enzyme onto a support has been employed to increase the loading of the enzyme of the carrier or change interactions of proteins with the surface. By this method, a surface layer is formed on an activated support, most often using an ionic polymer. An opposite charged layer is then deposited on top forming a thin layer by ionic attraction and the process is repeated until a desired thickness is achieved. Enzymes may be added between layers or as a final layer on the surface. Polymer brushes have been formed from polymeric surface by using free radical grafting. Glycidyl methacrylate, for example, has been grafted to hollow fiber polyethylene membranes for protein separation technologies by irradiation-induced free radical formation.
When used in a nonaqueous environment, immobilized enzymes can denature by being contact with a hydrophobic liquid or gas surface. To minimize interactions between a solvent or gas bubble, a hydrophilic polymer can be grafted as a thin layer over the enzyme/carrier to create a stable hydrophilic nanoenvironment. Coating of an aldehyde-activated dextrose over glucose oxidase immobilized to a non-porous carrier inhibited inactivation due to gas bubbles. Penicillian acylase was made more stable against dioxane by immobilizing the enzyme through multipoint bonding, followed by creating a hydrophilic environment on the carrier as well as on the enzyme, directly. If dextran was attached to just the carrier or just enzyme, no stabilization occurred indicating the necessity for complete coverage.
Use of spacer molecules such as glutaraldehyde, ethylenediamine, hexamethylene diamine, or poly(ethylene glycol) has been shown to provide an increase in the activity compared to conjugation directly to the support. Increasing the chain length aids in retaining enzymatic activity and the amino groups on a surface aids in the retention of D-amino oxidase activity when conjugated to a support. Likewise, studies in computational protein modeling at interfacial surfaces suggest that a hydrophilic chain on a hydrophobic surface would be necessary to preserve activity of an immobilized enzyme. Trypsin was separated from carboxylic-functionalized fleece by bovine serum albumin (BSA), aldehyde dextran, amino dextran, and PEG-diamine, and direct binding. Use of the spacer molecules showed an increase in activity in all cases relative to direct covalent binding. BSA showed the highest activity retention followed by amino dextran, PEG-diamine, and aldehyde dextran. Though the authors contribute enhance activity to decreased interaction of the protein with the hydrophobic fleece, charge may also be a factor since the surface of the fleece is charged, the aldehyde dextran is activated by periodiate oxidation of the dextran, and the amine spacers carry positive charges. Lactase was separated from an activated nylon support by diamines of varying chain lengths. Increased chain length resulted in an increased activity that was attributed to separation of the protein the densely charged surface. Distance may, also, promote mobility of the biocatalyst to decrease rigidity and allow for better dynamic motion for interaction of the protein with substrate.
Grafting and tethering of polymer chains onto a solid surface is used for increasing the available surface area for protein immobilization, to block activated functional groups on the surface from interaction with a substrate or fluid, and to provide a suitable surface for protein attachment or rejection. The nature of the polymer interface can be changed by grafting a polymer to the surface. Grafting and tethering provides a means of retaining the key bulk properties of a material while changing the surface for biointeractions. The choice of polymer at the surface can alter the interfacial thermodynamics and microenvironment since the enzyme will be reacting with the grafted layer, providing a more suitable platform for enzyme immobilization. Chitosan and collagen, for example, have been used to change the interface of hollow fiber membranes and nanofibers for lipase immobilization.
Self assembled monolayers (SAMs) are thin films of biological or chemical molecules that form spontaneously on surfaces. SAMs differ from graft in that the coverage formed by SAMs is an organized coverage consisting of a true monolayer, which may or may not be covalently grafted. Surface/substrates that form SAMS are limited—examples including alkanethiols on metal surfaces (particularly gold) and alkanesilanes on silicon. Alkanethiols have been shown to form a self assembled monolayer on the surface of gold by oxidative addition/reductive elimination. Alkanesilanes, likewise, self assemble on silicon oxide or silica glass. With respect to enzymes on carriers, SAMs with, different pendant groups, have been used to elucidate the mechanism for loss of glucose oxidase activity when adsorbed to a surface.
Multipoint covalent bonding is an alternative to surface modification and has been used to enhance enzyme stability in porous carriers by, three-dimensionally, fixing the enzyme in place so as to restrict movement and denaturation associated with entropy. Several enzymes have shown increased stability when applying this method. The activity retention associated with this method, which typically incorporates, a porous agarose carrier containing glycoxal residues, may be as low as 10-15%, or a high as 100%. Proper geometric alignment for the enzyme and the carrier is a limitation for this approach, and may lead to deactivation of the enzyme.
Physical Effects
The quaternary structure of an enzyme develops when subunits associate in a noncovalent manner. The association/disassociation of the subunits is dynamic, which can be problematic when conjugating to a planar surface where all the subunits may not contact the surface to be immobilized. Multipoint bonding in porous support if often effective for dimmers, but greater subunit associations may require a post-immobilization stabilization using an activated polymeric molecule. The quaternary structure of alcohol oxidases, with 4-8 subunits, were stabilized by a two step immobilization process of 1) attaching the enzyme to a porous support and 2) conjugating aldehyde-dextran to the enzyme/support. Though activity was reduced to 20%, quaternary structure (as determined by protein loss from the support in the presence of SDS and mercaptoethanol and analysis of the supernatant by electrophoresis) was maintained. In the same study, polyethylenimine (PEI) was used as a grafted layer, showing no loss of quaternary structure 50% activity retention when adsorbed to the support. The enzyme; however, retained more activity on the base agarose support (80%) prior to quaternary structure stabilization approaches. Tetrameric L-asparaginase was coupled to agarose-glutaraldehyde supports followed by conjugation with aldehyde-dextran. Then enzyme lost 60% of the intrinsic activity, but could be subjected to boiling SDS without loss of protein—demonstrating retention of the quaternary structure. Catalase from bovine liver on agarose beads showed full retention of activity; however, after washing, enzyme activity decreased and was attributed to loss of enzyme subunits in the rinse. Modification with aldehdye-dextran after immobilization caused a 30% reduction in enzymatic activity, but no loss of protein. Effects of washing on residual activity after immobilization; however, was not evaluated.
Crosslinking enzymes in solution to form carrier-free oligomeric microstructures are used to increase enzyme activity per unit area, provide enhanced thermostability and solvent stability, and stabilize quaternary structure. Cross-linked enzyme crystals (CLECs) are formed by cross-linking the crystallized structure of an enzyme to “freeze” the structure in place. Cross-linked enzyme aggregates, in a similar fashion, are formed by forcing enzymes to aggregate, by addition of salt or organic solvent, and cross-linking the structure. Glutaraldehyde is often used as the crosslinking agent because of its wide reactivity. CLECs/CLEAs can be used alone as an insoluble enzymatic platform or conjugated to a carrier. The limitations of CLEC/CLEAs are the small size, conditions for cross-linking, diffusion limitations, and deformation under mechanical stress. CLECs/CLEAs have been prepared for a number of enzymes, often resulting in greater stability under denaturing conditions. Chloroperoxidase, subilisin, theromolysin, and lipase have been crosslinked as CLECs or CLEAs. CLEAs of lipase have been immobilized in polymeric membranes to prevent leaching from the membrane without having to activate the support for covalent attachment. The process was carried out by allowing the enzyme to enter the membrane and then transferring the membrane to a solution of glutaraldehyde. Catalase, which is unstable in solution because of disassociation of the quaternary structure, was modified with glutaraldehyde—producing a stable conjugate. Though not an ideal CLEA, lactase has been aggregated on the surface of cotton by adding a PEI layer to the cotton, and not removing the excess PEI. When lactase is added, followed by glutaraldehyde, the lactase complexes in the PEI, and an aggregate PEI-lactase is formed.
Site directed orientation of an enzyme on a support can be useful in preventing non-specific adsorption of the protein and alignment of the active site towards the surface, and is particularly beneficial for enzymes acting on large polymeric substrates. Protein engineering has been used to introduce a cysteine residue on the surface of opposite the catalytic site to enhance to activity of immobilized subtilisin by rational design. A point mutation is achieved by substituting the codon calling for a cysteine residue at the desired place in the gene sequence, and introducing the gene into an expression vector to produce the new protein. His-tagged enzymes are proteins that have genetically engineered by introducing a sequence calling for histidine residues (usually six) at the C- or N-terminus. The histidine sequence reacts specifically with divalent metal cations. By covalently attaching a metal chelating agent like Ni-nitriloacetic acid (NTA), and introducing Ni++, a site-specific interaction will form between the chelating agent, nickel, and the histidine sequence. Polyhydroxyalkanoate was immobilized by this method to silicon for the production of aliphatic polyesters. Another example of site-specific immobilization is that of cutinase, a serine esterase, which forms a covalent bond with phosphonate inhibitors and is specific to this enzyme. Some enzymes are glycosylated and can be conjugated to a support specifically by the carbohydrate moiety. Lipase has been immobilized in this fashion by periodate oxidation of the carbodydrate chain for conjugation to Eupergit C supports. Taking advantage of the pKa difference in the N-terminus has been used to site-specifically attach proteins to PEG. General bioconjugation schemes, targeting the same amino acid group can, likewise, result in different retention in enzyme activity. This phenomenon may be due to the specificity of the bioconjugation reagent with the desired amino acid groups.
Reducing the size of the carrier to the nanoscale has shown to promote increased activity of an immobilized enzyme, due to either increased Brownian motion or a reduction in protein conformational changes upon immobilization The effect of particle size was demonstrated using α-Chymotrypsin attached to polystyrene particles of 100-1000 nm as well as thin films of polystyrene. Though the change was slight for the nanoparticles, kcat/Km was 100 fold lower on the films, which the authors attributed to mobility effects of the catalyst Inhibition of enzyme conformational changes has, also, been attributed to the advantageous geometry of nanomaterials. Soybean peroxidase deactivation was measured after adsorption to single walled nanotubes (SWNTs) and graphite flakes. The deactivation kinetics of the enzyme was lower on the SWNTs and demonstrated to be independent of protein coverage, while the graphite flakes were dependent on protein coverage. It was concluded that denaturing lateral protein-protein interactions were decreased due to the size and curvature of the SWNTs.
The porosity of a carrier can effect apparent enzyme activity by increasing Km of an enzyme in a three dimensional medium. Diffusion limitations of the substrate can be effected by the microenvironment of the carrier/solvent, inaccessibility of substrate into the pores, or blockage of available enzyme. Likewise, the microenvironmental pH can effect how the substrate interacts with the carrier-bound enzyme. Trypsin showed a 2-unit pH shift for activity when immobilized in a carboxylic acid matrix.
Ordering of the protein on the surface can alter the activity of the conjugated enzymes. Crowding of protein on the surface of a support with increase loading has been shown to reduce the activity of the enzyme due spatial restrictions, limited active site accessibility, or denaturing of the protein. Polymethylmethacrylate (PMMA) with an irregular surface compose of it-PMMA or an order surface of alternating it- and st-PMMA (which forms regular helical structures) was used to study immobilization of lactase (E. coli). The PMMA with the ordered surface resulted in the retention of greater lactase activity. This result was attributed to enthalpic gains associated with polymer mobility, and weak/limited interactions between the enzyme and alternating surface that limited these enthalpic gains.Carrier and Enzyme ModificationSurface Modification
Functionalization of materials by copolymerization, wet chemistry, and physical-chemical methods are employed to change the characteristics of a surface or develop a surface more suitable for bioconjugation. Introduction by copolymerization insert a monomer with an inert side chain to be polymerized with a polymer containing a functional group. Polystyrene microspheres have been successfully prepared using carboxylic acid, amine, and hydroxyl monomers. Wet chemistry techniques make use of chemical groups in the polymer side chain (i.e. hydroxyls, esters, amines, aldehydes, and carboxylic acids) or by introducing groups for an inert surface such as oxidation or nitrosylation. Physical techniques such as oxygen and nitrogen plasma oxidation, corona, and UV treatment have, also, been instrumental in modifying a polymer surface and are used in a number of industries including food applications. These methods have provided a means to conjugate bioactive compounds to polymeric supports.
Enzyme Modification
Biological methods of enzyme modification are focused on manipulation of the enzyme at the genetic level. Transformations in the gene and corresponding protein structure are either random (directed evolution) or intentional (rational design). Directed evolution relies on methods such as error-prone PCR and DNA shuffling. epPCR utilizes polymerase to introduce random mutations in a gene. DNA shuffling uses a number of homologous genes with desired characteristics. The genes are fragmented, denatured, and annealed in random fashion. For both epPCR and DNA shuffling, the new genes are introduced in an expression vector and the corresponding protein screened for a desired property. Directed evolution has produced a number of enzymes with enhanced specificity, activity, and thermostability. The method, though quick and powerful, requires large libraries of genes and multiple generations of protein producing microorganisms with no guarantee of enzyme improvement. Rational design is a technique where an intentional modification is introduced in the sequence of the gene to alter the protein. The practice aims to go from desired function to structure to sequence. Compared to directed evolution, rational design has fewer successes, attributed to the idea that knowledge of structure function of enzyme is still developing.
Chemistry-driven modifications are employed to change the surface interactions between the enzyme and the external environment, adjust enantioselectivity and/or specificity, prevent unwanted interactions, or explore structure/function relationships. Engineering in this category include the use of surfactants, reverse micelles, extraction, and covalent modification. Surfactants may be used by directly incorporating a lyophilized protein into a solvent containing surfactant and small amounts of water. The continuous solution is then centrifuged and the supernatant removed. This method has shown 25-72% solubility of enzymes in a number of solvents of varying polarity as well as protein aggregation of up to 100 molecules.
Reverse micelle containing enzyme are produced by dissolving an aqueous solution of enzyme in an organic solvent containing a surfactant. The self-assembled micelles encapsulate enzyme and can be separated by centrifugation. This method allows some water to be associated with the system, which may increase the unfolding of the enzyme. Protein extraction is a unique techniques that involves ion-pairing an enzyme with a small molecule, which can be paired with a surfactant molecule and extracted into an organic layer. The method has been used with up to 95% solubilization of an enzyme, and can be applied in crosslinking an enzyme during monomer polymerization.
Polymer conjugation may be used to make the enzyme surface more hydrophilic or hydrophobic to induce solubility or thermostability. This technique utilizes single polymer chains or small molecules to attach to the surface of the enzyme. The most commonly polymeric molecules for covalent modification are carbohydrates (i.e., cyclodextrans, pectin, chitosan, carboxymethylcellulose, and sucrose) and poly(ethylene glycol). A procedure of interest for that has been utilized for the enhancement of therapeutic proteins and peptides is PEGylation, which unlike many other modifications provides simple, clean, and specific attachment chemistry for protein derivatives with low toxicity. PEGylation involves the attachment of polyethylene glycol (PEG) to a functional group of a compound so as to induce a variety of alterations to the protein including decreased immunogenicy, increased half-life due to reduced proteolysis, increased thermostability, and alterations in the solubility properties, which allows the protein to be soluble in water, toluene, 1,1,1 trichloroehane and benzene, and insoluble in ethyl ether. Factors influencing these properties are the number of PEG molecules attached, the molecular weight and structure of PEG chains attached, the location of PEG sites on the protein, and the chemistry used.
The solubility of enzymes in organic solvents is enhanced upon attachment of PEG, and PEGylation nearly always increased solubility. An increase in benzene solubility of catalase is also accompanied with increasing degree of PEG-modification. There is indication that PEG-modified enzymes catalyze reactions in hydrophobic media and, in some cases, affinity for the substrate and velocities are increased in such, but there is little evidence to suggest whether this increase is due to stabilizing effects of PEG, solubilization that increases protein-substrate interaction, or a synergistic effect. The thermostability of effects of PEG on protein stabilization has been well documented, with a number of enzymes having demonstrated an increased thermostability after being modified with PEG. A detailed study on the thermostability of three enzymes after modification by glycosalation and by PEGylation showed that glycosylation increased hydrophilicity of the enzymes while PEG made the enzyme more hydrophobic. Under heat denaturing conditions, the glycosylated enzymes lost more activity over time compared to the native enzymes, but PEGylated enzymes showed an increased stability over time. These results were attributed to the ability of PEG to draw water from the system to be utilized by the shell, but also increasing interaction of water with hydrophobic clusters, preventing access to the enzyme surface. Other studies, however, have shown that an increase in enzyme thermostability upon modification with polysaccharides, though similar mechanisms for enzyme stability may be attributed to these modifications as well.
Small molecules have been used to study structure function relationships of enzymes and to alter the stability of the proteins. Molecules are often attached to thiol or ionic groups of the enzyme because of the reactivity; however, modifications of these groups may lead to inactivation of the enzyme. Chymotrypsin has been glycosylated to promote stable oligomeric structures of the enzyme. The enzyme, RNase A, has also been glycosylated with glucosamine to explore thermostability of the enzyme. Immobilized lipase has been aminated with EDC/ethylenediamine to change the selectivity of the enzyme. Though selectivity change, intrinsic activity was reduced by 70%. The amine and carboxylic acid groups of penicillin G acylase were converted to opposite charge by amination of the carboxylic acid groups with ethylenediamine or succinylation of the amine groups with succinic anhydride. The modifications were performed to increase loading of the enzyme on ion exchange resins without dramatic changes to enzyme activity.